福氏2a志贺氏菌glpR、deoR基因功能的研究
发布时间：2019-04-19 07:19
【摘要】： 志贺氏菌属(Shigella spp.)细菌是一类不形成芽孢的革兰氏阴性致病菌,能够通过侵袭大肠引起患者典型菌痢症状(发热、腹痛、腹泻)。根据生化反应和O-抗原结构的不同,志贺氏菌可以分为四个群。其中,福氏志贺氏菌和宋内志贺氏菌比鲍氏志贺氏菌和痢疾志贺氏菌分布更广。据WHO统计,每年约有1.647亿人感染痢疾,110万人死亡,大多数为5岁以下的儿童。 福氏志贺氏菌(S. flexneri)是发展中国家主要的感染菌株,2a血清是主要的血清型。近几年来我国每年大约有2000万人次感染痢疾,年累计发病数一直位于第3位,仅次于肺结核和乙肝。由于志贺氏菌感染剂量极低(10-100个细菌),主要传播途径为粪口途径,所以最常见的扩散形式是人与人之间的传播,加之多重耐药菌株的不断出现,常规的抗生素治疗方式遇到了巨大的挑战。由于人们对痢疾杆菌的致病机理和宿主的免疫保护机制还不十分清楚,所以迄今为止仍未研究出能有效控制痢疾的理想疫苗。 有研究表明,福氏2a志贺氏菌在37℃时发挥完整的细胞侵袭力,而在30℃时却不能侵袭上皮细胞。本实验室对福氏2a志贺氏菌2457T不同培养温度下全菌蛋白的比较蛋白质组研究发现:当温度升高到37℃时,GlpQ、GlpB蛋白表达上调。而当福氏2a志贺氏菌2457T毒力大质粒缺失时,GlpQ、GlpB蛋白表达也显著上调。显然,GlpQ、GlpB蛋白对福氏2a志贺氏菌2457T的毒力有影响。而GlpQ、GlpB蛋白由GlpR、DeoR、H-NS三个蛋白调控,H-NS蛋白的研究已有报道,后边也有讨论,而GlpR、DeoR蛋白属于DeoR蛋白家族,它们是一种细菌性调节蛋白(PF00455)。DeoR是一种转录调节子,阻遏脱氧核糖的代谢。GlpR也是一种转录调节子,调节3-磷酸甘油的阻遏。 为了探索福氏2a志贺氏菌2457T中glpR、deoR基因的功能,本研究将由Datsenko和Wanner建立的λ噬菌体Red重组系统稍加改进,成功地敲除了福氏2a志贺氏菌2457T株的glpR、deoR基因,并利用低拷贝质粒构建了回复体加以验证,对GlpR、DeoR进行了初步的功能研究。对野生株、突变株与回复株的生长曲线、生化反应进行了比较分析,结果表明,野生株、突变株和回复株的生长曲线基本一致;突变株与野生株的生化反应没有明显差异。通过豚鼠角膜实验、HeLa细胞模型和小鼠动物模型检测glpR、deoR基因缺失对志贺氏菌毒力的影响,发现野生株、突变株与回复株均能够引起豚鼠角膜强烈炎症反应,而glpR基因的缺失会明显提高志贺氏菌的毒力。利用双向电泳和质谱技术对野生株、突变株和回复株进行了全菌蛋白的比较蛋白质组学研究。在蛋白质组的比较分析中,我们发现,glpR、deoR基因敲除和回复后,表达上调的蛋白点共有14个,表达下调的有3个。结果表明,由于glpR、deoR基因都是转录抑制子,它们的缺失,能使其抑制的一系列蛋白表达上调。缺失glpR基因后,甘油激酶(GlpK)、3-磷酸甘油脱氢酶B亚单位(GlpB)和磷酸二酯酶(GlpQ)表达上调。而缺失deoR后,胸苷磷酸化酶(DeoA)、嘌呤核苷磷酸(DeoD)、2 -脱氧核糖- 5 -磷酸醛缩酶(DeoC)等表达上调。 YciD (NP-836950)是含有212个氨基酸残基的,分子量为22kDa的小分子量假想外膜蛋白,属于OmpW家族蛋白,该蛋白的功能未知。本实验室在福氏2a志贺菌2457T野生株与hns(又称virR)基因缺失突变株蛋白表达谱的比较研究中发现,37℃培养条件下,hns基因缺失突变株中外膜蛋白YciD表达量下调,我们认为该蛋白可能受H-NS的调控。为探索福氏2a志贺菌2457T中yciD基因的功能,本文采用改进的λ-Red重组系统成功构建了该基因的缺失突变体。
[Abstract]:Shigella spp. ) The bacteria are a class of Gram-negative pathogenic bacteria that do not form a spore, and can cause typical bacillary dysentery (fever, abdominal pain, and diarrhea) caused by the invasion of the large intestine. Shigella can be divided into four groups according to the biochemical reaction and the O-antigen structure. Among them, Shigella flexneri and Shigella sonneae are more widely distributed than Shigella flexneri and Shigella in Shigella. According to the WHO statistics, about 164.7 million people are infected with diarrhea each year, and 1.1 million people die, and most of them are children under the age of 5. Shigella flexneri (S. flexneri) is the main infection strain in developing countries, and 2a serum is the main Serotype. In recent years, about 20 million people have been infected with dysentery per year, and the cumulative incidence of the year has been in the third place, second only to tuberculosis. As a result of the extremely low dose of Shigella (10-100 bacteria), the main transmission route is the faecal route, so the most common form of diffusion is the spread of human and human, and the continuous appearance of multiple drug-resistant strains, and the conventional antibiotic treatment method has encountered a great deal. The challenge is that the mechanism of the disease and the mechanism of the immune protection of the host are not very clear so far, the reason for the effective control of the dysentery has not been studied so far The results of the study showed that the 2-a-shigella flexneri exhibited a complete cell-invasion force at 37 & deg; C, while at 30 鈩，
本文编号：2460730
[Abstract]:Shigella spp. ) The bacteria are a class of Gram-negative pathogenic bacteria that do not form a spore, and can cause typical bacillary dysentery (fever, abdominal pain, and diarrhea) caused by the invasion of the large intestine. Shigella can be divided into four groups according to the biochemical reaction and the O-antigen structure. Among them, Shigella flexneri and Shigella sonneae are more widely distributed than Shigella flexneri and Shigella in Shigella. According to the WHO statistics, about 164.7 million people are infected with diarrhea each year, and 1.1 million people die, and most of them are children under the age of 5. Shigella flexneri (S. flexneri) is the main infection strain in developing countries, and 2a serum is the main Serotype. In recent years, about 20 million people have been infected with dysentery per year, and the cumulative incidence of the year has been in the third place, second only to tuberculosis. As a result of the extremely low dose of Shigella (10-100 bacteria), the main transmission route is the faecal route, so the most common form of diffusion is the spread of human and human, and the continuous appearance of multiple drug-resistant strains, and the conventional antibiotic treatment method has encountered a great deal. The challenge is that the mechanism of the disease and the mechanism of the immune protection of the host are not very clear so far, the reason for the effective control of the dysentery has not been studied so far The results of the study showed that the 2-a-shigella flexneri exhibited a complete cell-invasion force at 37 & deg; C, while at 30 鈩，
本文编号：2460730
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